Cold stress and chilling injury in plants

To understand how plants cope with chilling stress, it is essential to know the molecules and pathways involved in the chilling tolerance or sensitivity in tomato fruit. Chilling stress is important during storage at low temperature, which is an appropriate strategy to prolong the market life of many vegetables [Kader, 2003]. This practice drops the functioning of metabolic pathways and reduces pathogenic events, making exportation more favorable for long distance shipment and consequently, a more regulated supply of fruit in the market arises. However, the outcome of storing the plant products at low, critical temperatures is chilling injury (CI) that generates high economic losses. Sensitive to low temperatures are not only fruits, but vegetables, and ornamentals of tropical or subtropical origin. Certain horticultural crops of temperate origin are also susceptible to chilling injury such as tomato. These temperate crops, in general, have lower threshold temperatures, around 5 °C [Wang, 2004a]. CI is the set of physiological alterations and dysfunction that appear during the exposure of plants to low temperatures above the freezing point (between 0 ºC and 15 ºC) [Lyons, 1973]. Many

physiological and biochemical consequences of chilling injury have been widely described.

However, the molecular mechanisms behind the generation and tolerance to chilling stress in fruit remain to be understood.

Under chilling temperatures, tissues deteriorate because they are incapable of carrying on regular metabolic processes. Chilling-sensitive species respond to chilling stress by altering numerous processes at physiological and biochemical levels and thus, the cells start to function anomaly [Wang, 2004a]. These disorders constitute one of the main limitations of the commercial life of many fruit and vegetable products. Reducing the effects of CI would lead to greater availability of food, smaller areas of land needed for cultivation and the possibility of exporting to new international markets.

1.4.1. Symptoms of CI.

In fruit, symptoms of CI are diverse and depend mainly on the cultivar, the temperature and time of exposure, the degree of maturity, the climatic characteristics of the growing area and the temperatures prior harvesting. Other factors can also affect the postharvest life of cold stored fruits and the development of CI, such as the relative humidity of the environment and the presence of ethylene in the storage atmosphere. Common symptoms of CI in fruits include depressions and surface wounds, pitting, internal colour alterations, water-soaking of the tissue, inability to typically ripen which causes lack of uniformity in the surface and pulp colour, higher susceptibility to microorganisms and pathogens and loss of water [Lyons, 1973; Wang, 2010]. Besides, there is a decrease in the sweetness, aroma and characteristic flavor of the fruit, caused by a metabolic imbalance [Maul, 2000]. Surface pitting is one of the most common symptoms in many fruits and vegetables such as citrus fruits, cucumbers, eggplant, melons, and sweet potatoes. Failure to ripen was observed in chilled avocados, bananas, mangos, melons, and tomatoes. Also, it is usually the internal discoloration in avocados, pineapples, and sweet potatoes [Wang, 2010]. This diversity of CI symptoms of tropical and subtropical fruits and vegetables suggests multiple responses to low temperature. Products that are stored at chilling temperatures do not show CI symptoms when remaining in low temperatures. In some cases, these characteristics can develop progressively during the exposure to low temperatures but usually develop and become evident in a short time after products are transferred to room temperature [Malacrida, 2006; Gonzalez, 2015].

1.4.2. The theories of CI.

In previews reports, some molecular mechanisms have been suggested to accommodate the physiological and biochemical changes associated with CI [Lukatkin, 2012]. One of the most discussed theory proposed that chilling sensitivity can be explained by a phase transition of cell membranes that occurs at low temperatures and converts the membranes from a flexible liquid-crystal state into a rigid state of gel-solid. These changes trigger severe alterations at the membrane and enzymatic membrane-binding system that culminate with adverse events such as membrane damage, loss of electrolytes, failure of respiration and the production of toxic compounds. This phase transition in even a small proportion of membrane lipids results in the formation of solid domains that can cause cell damage. When the exposure to cold is brief, the effect may be transient, and the cell survives. However, when stress is prolonged, necrosis and cell death occur [Lyons, 1973]. This hypothesis that proposed the phase transition of membrane lipids as the primary cause of CI has been discredited. The membrane changes do not happen instantly after the start of chilling and are more likely to be less critical syndromes. According to the phase transition hypothesis, the rise in membrane permeability occurring because of the low-temperature condition (causing membranes leakage) should be fast, recorded immediately (minute scale) after placing the tissue at chilling temperatures.

Nevertheless, this is not observed, and frequently passive permeability is not augmented.

However, there is no doubt that the physical properties of membranes are crucial for cell homeostasis, and that they may be especially deteriorated during the cellular response to chilling stress [Lukatkin, 2012].

Another theory of CI is based on the metabolic disorder that occurs in cold temperature. In this case, cell death takes place due to the prevalence of disintegration over synthesis, and to the dissociation of enzymes and other proteins, which would result in changes in enzymatic kinetics and/or in structural changes of specific proteins such as tubulins [Graham, 1982]. Low temperatures would induce a decrease of hydrophobic binding forces, altering protein-protein and lipid-protein interactions. The overall disruption may cause the malfunction of soluble enzymes, dissociation of subunits and unfolding of the proteins [Parkin, 1989].

In other reports, special attention has been drawn to two different hypotheses to explain the induction of CI, one to a fast uprise in the free cytosolic Ca2+ level ([Ca2+]cyt) and the other to the occurrence of oxidative stress upon chilling [Minorsky, 1985; Prasad, 1994]. The quick rise in [Ca2+]cyt due to chilling, may work for as the primary physiological indication of cold exposure. Changes in [Ca2+]cyt activate cascade reactions in the cell, which leads to many disorders at all levels of organization. When changes in the compartmentation of calcium in the chilled plants occurred, they directed to an increase in [Ca2+]cyt, end cytoplasmic streaming and disturb the subcellular structures. For proposing the calcium hypothesis, it was taking into

account the oxidative stress during chilling that plays a crucial function in the transduction of the chilling signal. It was shown that [Ca2+]cyt changes and oxidative stress are intimately connected under chilling conditions. The increase of free radicals and ROS in chilling produce substantial alteration to membrane lipids and other cellular components [Suzuki, 2006].

1.4.3. CI in tomato fruit.

The effect of cold storage in the ripening process of tomato fruit has been the subject of numerous studies [Rugkong, 2011; Ré, 2012; Tao, 2014; Cruz-Mendívil, 2015; Gonzalez, 2015]. The global transcriptomic analysis in cold-stored tomato fruit revealed the down-regulation of genes involved in color development, including phytoenesynthase1 and carotenoid isomerase, and in genes encoding the cell wall modifying proteins polygalacturonase, pectin esterase1, β-galactosidase, expansin1, and xyloglucan endotransglucosylase-hydrolase 5 [Rugkong, 2011]. Besides the reduction of color-related genes, the alteration described in the coloration of tomato fruit was explained by the inability to accumulate lycopene [Watkins, 1990; Malacrida, 2006]. To understand the mechanisms responsible for the tolerance to CI observed in Micro-Tom fruit, a recent study combined metabolomics and transcriptomics data of Micro-Tom fruit after chilling storage and the changes in the overall metabolome including primary metabolites, carotenoids, lycopene, soluble antioxidants, tocopherols, and tocotrienols, and transcriptome after chilling were investigated. The results showed alterations in the metabolism of reserves, fermentation and amino acids mobilization and photosynthesis, and the induction of defense mechanisms. After removing the fruit from refrigeration, the photosynthetic activities and the transcripts related showed a minor recovery. Transcriptional up-regulation of genes coding for proteins that accumulate in response to low temperatures, along with genes encoding antioxidant enzymes and sHSP was also observed. In addition to this, it was found a robust up-regulation of AOX gene transcription and a rise in pyruvate content which is a positive effector of AOX. Moreover, the level of ethanol and several genes involved in fermentation processes increased after chilling indicating a fermentative physiological response of the fruit [Gonzalez, 2019].

Proteomics studies have shown that the levels of proteins related to maturation of the fruit decreased with cold storage, while proteins related to the stress response increased [Page, 2010]. It has been shown that storage at low temperatures also decreases respiration and ethylene synthesis, what could modify the expression of many genes that explain the following symptoms, although it has also been seen that ethylene is not essential for the appearance of symptoms of CI [Luengwilai, 2010].

1.4.4. Oxidative stress and CI.

All aerobic organisms require oxygen as an essential element of their metabolism. The oxygenic environment may, however, involve potential hazards for the cells. During normal metabolism, intermediary products of the reduction of oxygen that show a high reactivity are produced in various subcellular compartments. These intermediates are called ROS [Mittler, 2002]. ROS species include superoxide anion (O2^-), hydrogen peroxide (H2O2), hydroxyl radical (^.OH) and singlet oxygen (¹O2) produced by physical excitation of O2. In the course of evolution, plants have developed an intricate and efficient network to remove and mitigate the toxicity of ROS and started to use some of these toxic molecules as mediators in signal transduction [Mittler, 2004; Bailey-Serres, 2006]. It has been proposed that ROS action has a double effect in plants, acting as toxic compounds, and at the same time as crucial regulators of numerous pathways related with critical biological processes such as growth, cell cycle, programmed cell death, hormonal signalling, response to biotic and abiotic stress, and development [Mittler, 2004]. To maintain the dual role, there must be a delicate balance between the production and the removal of ROS. The term "oxidative stress" is usually used to describe situations in which the generation of ROS surpasses the cell capacity of keeping the redox homeostasis [Gill, 2010].

In plants, the production of ROS occurs in the apoplast and several subcellular compartments such as peroxisomes, chloroplasts, mitochondria and the nucleus [Toivonen, 2004]. Although chloroplasts are usually the primary site of ROS production in plants, in post-harvest fruit other organelles may become important sites for the generation of ROS. Due to their high oxygen consumption, mitochondria are the leading producers of ROS in non-photosynthetic tissues [Hodges, 2003].

ROS molecules can react producing lipid peroxidation, polysaccharides and protein degradation, and disruption of DNA molecules. When the intracellular ROS concentration increases uncontrollably, irreversible damage is produced leading to cell death [Gill, 2010]. A model has been proposed to describe the function of ROS signaling pathways in the chilling stress response. According to this model, CI probably start from a membrane receptor, still unknown, that would sense the change in the temperature and activate an NADPH membrane oxidase, causing a controlled increase in the levels of ROS, which would function as a regulatory signal for the expression of responsive genes [Einset, 2007].

The oxidative stress produced during storage at low temperatures is one of the main factors that contribute to the generation of CI. The loss of integrity and fluidity of membranes may affect the protein functions either by the direct action of ROS, by alteration of the activity due to an unfavorable lipid context or by the conjunction of both situations. In this way, the alteration and dysfunction of critical enzymes lead to a metabolic imbalance that manifests at the tissue

level as a visible symptom of CI [Prasad, 1994; Shewfelt, 2000]. Many studies have linked the occurrence of post-harvest CI after storage of fruit at low temperatures with the generation of oxidative stress, and it has been postulated that the antioxidant capacity of the cell may play an essential role in the prevention of CI symptoms. It has been shown that cold-tolerant species produce fewer ROSs and more antioxidant compounds [Sala, 1999; Malacrida, 2006].